Role of interferon-gamma release assay for screening and monitoring of latent tuberculosis infection in kidney transplant recipients.

BACKGROUND: The reactivation of tuberculosis (TB) among kidney transplant (KT) recipients in an endemic area is of general concern. However, the epidemiology of latent TB infection (LTBI) status and its dynamic change responses have not been explored. METHODS: Between September 2020 and August 2021, a prospective study was conducted to investigate the status of LTBI in KT recipients who received a 9-month isoniazid universal prophylaxis. This status was measured using the interferon-gamma release assay (IGRA) with T-SPOT.TB before transplant, as well as at one month and nine months post-transplant. RESULTS: Ninety-one KT recipients had a mean (SD) age of 45 (11) years, and 41% were female. Sixty-eight (75%) patients received a deceased donor allograft, and eighty-six (91%) patients received induction immunosuppressive therapy. The IGRA results were positive, borderline, negative, and indeterminate in 14 (15.4%), 6 (6.6%), 64 (70.3%), and 7 (7.8%) patients, respectively. Among 84 evaluable patients, 20 (23.8%) KT recipients were defined as having LTBI. Older age was significantly associated with LTBI (OR 1.06 [95% CI 1.01-1.12], p = 0.03). Among the 77 KT recipients who completed monitoring, 55 had negative IGRA results. Three (5.4%) KT recipients had conversion post-transplant. One of them developed pulmonary TB at 1 week after the transplant. Among the 13 patients with positive results, 8 (61.5%) remained positive, 1 (7.7%) had an indeterminate result at 1-month post-transplant and subsequently tested positive at 9 months post-transplant, and 4 (30.8%) experienced reversion to negative results throughout the study. CONCLUSIONS: In a high TB-endemic area, one-quarter of KT recipients were reported to have LTBI, and the dynamic change of IGRA response in KT recipients is plausible post-transplant.

T Cell Peptide Prediction, Immune Response, and Host-Pathogen Relationship in Vaccinated and Recovered from Mild COVID-19 Subjects.

COVID-19 remains a significant threat, particularly to vulnerable populations. The emergence of new variants necessitates the development of treatments and vaccines that induce both humoral and cellular immunity. This study aimed to identify potentially immunogenic SARS-CoV-2 peptides and to explore the intricate host-pathogen interactions involving peripheral immune responses, memory profiles, and various demographic, clinical, and lifestyle factors. Using in silico and experimental methods, we identified several CD8-restricted SARS-CoV-2 peptides that are either poorly studied or have previously unreported immunogenicity: fifteen from the Spike and three each from non-structural proteins Nsp1-2-3-16. A Spike peptide, LA-9, demonstrated a 57% response rate in ELISpot assays using PBMCs from 14 HLA-A*02:01 positive, vaccinated, and mild-COVID-19 recovered subjects, indicating its potential for diagnostics, research, and multi-epitope vaccine platforms. We also found that younger individuals, with fewer vaccine doses and longer intervals since infection, showed lower anti-Spike (ELISA) and anti-Wuhan neutralizing antibodies (pseudovirus assay), higher naïve T cells, and lower central memory, effector memory, and CD4hiCD8low T cells (flow cytometry) compared to older subjects. In our cohort, a higher prevalence of Vδ2-γδ and DN T cells, and fewer naïve CD8 T cells, seemed to correlate with strong cellular and lower anti-NP antibody responses and to associate with Omicron infection, absence of confusional state, and habitual sporting activity.

Validation of the Enzyme-Linked ImmunoSpot Analytic Method for the Detection of Human IFN-γ from Peripheral Blood Mononuclear Cells in Response to the SARS-CoV-2 Spike Protein.

COVID-19 vaccine evaluations are mainly focused on antibody analyses, but there is growing interest in measuring the cellular immune responses from the researchers evaluating these vaccines. The cellular responses to several COVID-19 vaccines have been studied using the enzyme-linked immunospot (ELISPOT) assay for IFN-γ. However, the ELISPOT assay is no longer used only for research purpose and so the performance of this assay must be validated. Since the bioanalytical validation of ELISPOT-IFN-γ is essential for evaluating the method's effectiveness and establishing confidence in a vaccine's immunogenicity, the present work validates the ELISPOT-IFN-γ assay's performance in determining the frequency of IFN-γ-producing cells after stimulation with the SARS-CoV-2 spike protein. The validation was performed in peripheral blood mononuclear cells from volunteers immunized with anti-COVID-19 vaccines. According to the findings, the LOD was 17 SFU and the LLOQ was 22 SFU, which makes the method highly sensitive and suitable for evaluating low levels of cellular responses. The procedure's accuracy is confirmed by the correlation coefficients for the spike protein and anti-CD3+, being 0.98 and 0.95, respectively. The repeatability and intermediate precision tests were confirmed to be reliable by obtaining a coefficient of variation of ≤25%. The results obtained in this validation enable the assay to be employed for studying antigen-specific cells and evaluating cellular responses to vaccines.

Identification of Candidate Immunodominant Epitopes and Their HLA-Binding Prediction on BK Polyomavirus Proteins in Healthy Donors.

BK polyomavirus infection is an important cause of graft loss in transplant patients, however, currently available therapies lack effectiveness against this pathogen. Identification of immunological targets for potential treatments is therefore necessary. The aim of this study was to predict candidates of immunodominant epitopes within four BK virus proteins (VP1, VP2, VP3 and LTA) using PBMCs from 44 healthy donors. We used the ELISpot epitope mapping method to evaluate the T-cell response, and HLA-peptide binding was predicted using the NetMHCpan algorithm. A total of 11 potential peptides were selected for VP1, 3 for VP2/VP3 and 13 for LTA. Greater reactivity was observed for VP1 and LTA proteins compared with VP2/VP3. Most of the peptides selected as potential immunodominant candidates were restricted towards several HLA class I and II alleles, with predominant HLA class I binding by computational predictions. Based on these findings, the sequences of the selected immunodominant epitopes candidates and their corresponding HLA restrictions could contribute to the optimisation of functional assays and aid in the design and improvement of immunotherapy strategies against BK virus infections.

High prevalence of long COVID in anti-TPO positive euthyroid individuals with strongly elevated SARS-CoV-2-specific T cell responses and moderately raised anti-spike IgG levels 23 months post-infection.

Introduction: Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) infection, the causative agent of coronavirus disease 2019 (COVID-19), causes post-acute infection syndrome in a surprisingly large number of cases worldwide. This condition, also known as long COVID or post-acute sequelae of COVID-19, is characterized by extremely complex symptoms and pathology. There is a growing consensus that this condition is a consequence of virus-induced immune activation and the inflammatory cascade, with its prolonged duration caused by a persistent virus reservoir. Methods: In this cross-sectional study, we analyzed the SARS-CoV-2-specific T cell response against the spike, nucleocapsid, and membrane proteins, as well as the levels of spike-specific IgG antibodies in 51 healthcare workers, categorized into long COVID or convalescent control groups based on the presence or absence of post-acute symptoms. Additionally, we compared the levels of autoantibodies previously identified during acute or critical COVID-19, including anti-dsDNA, anti-cardiolipin, anti-ß2-glycoprotein I, anti-neutrophil cytoplasmic antibodies, and anti-thyroid peroxidase (anti-TPO). Furthermore, we analyzed the antibody levels targeting six nuclear antigens within the ENA-6 S panel, as positivity for certain anti-nuclear antibodies has recently been shown to associate not only with acute COVID-19 but also with long COVID. Finally, we examined the frequency of diabetes in both groups. Our investigations were conducted at an average of 18.2 months (convalescent control group) and 23.1 months (long COVID group) after confirmed acute COVID-19 infection, and an average of 21 months after booster vaccination. Results: Our results showed significant differences between the two groups regarding the occurrence of acute infection relative to administering the individual vaccine doses, the frequency of acute symptoms, and the T cell response against all structural SARS-CoV-2 proteins. A statistical association was observed between the incidence of long COVID symptoms and highly elevated anti-TPO antibodies based on Pearson's chi-squared test. Although patients with long COVID showed moderately elevated anti-SARS-CoV-2 spike IgG serum antibody levels compared to control participants, and further differences were found regarding the positivity for anti-nuclear antibodies, anti-dsDNA, and HbA1c levels between the two groups, these differences were not statistically significant. Disscussion: This study highlights the need for close monitoring of long COVID development in patients with elevated anti-TPO titers, which can be indicated by strongly elevated SARS-CoV-2-specific T cell response and moderately raised anti-spike IgG levels even long after the acute infection. However, our results do not exclude the possibility of new-onset thyroid autoimmunity after COVID-19, and further investigations are required to clarify the etiological link between highly elevated anti-TPO titers and long COVID.

Measurement of the Memory B Cell Response via Antibodies from Activated Cells.

BACKGROUND/OBJECTIVES: The body's immune response to infections and vaccination leads to the formation of memory B cells (MBCs), which protect against future infections. MBCs circulate in the blood, and the strength of the MBC response is measured with different tests. In this study, tests to measure the MBC response were compared. METHODS: An MBC enzyme-linked immunospot assay (MBC-ELISpot), which counts the antibody-releasing cells (MASCs) that arise from memory B cells in vitro, and two versions of an MBC enzyme-linked immunosorbent assay (MBC-ELISA), which measures the concentration of antibodies released by the MASCs, were compared. The lower measurement limit of MBC-ELISpot and ELISA was determined, and it was investigated how the measurement results of individual samples and in a sample of test persons correlate. RESULTS: Both methods had similar lower limits of detection, and the antibody concentration correlated strongly with the number of MASCs in individual samples. The antibody concentrations measured on a sample correlated with Pearson correlation coefficients of R = 0.83-0.87 with the number of MASCs, and the proportion of antigen-specific antibodies in total IgG correlated with R = 0.74-0.82 with the proportion of antigen-specific MASCs in all antibody-secreting cells. CONCLUSIONS: Since the measurement sensitivity of MBC-ELISA and MBC-ELISpot is similar and the measurement results correlate strongly in a random sample, the tests are interchangeable. The MBC-ELISA has an advantage over the ELISpot in that the antibody measurements can be divided up over time, repeated, and extended. This creates new possibilities for measuring the MBC response.

Decreased variability in the site-specific results during participation in the external quality assurance program oversight laboratory (EQAPOL) proficiency program for IFN-gamma enzyme-linked immunospot (IFN-γ ELISpot) assay.

The NIAID DAIDS-sponsored External Quality Assurance Program Oversight Laboratory (EQAPOL) manages an interferon-gamma (IFN-γ) enzyme-linked immunospot (ELISpot) external proficiency program. The ELISpot program evaluates the accuracy and variability of results across laboratories. The variability in the program is quantified via the dispersion, which is the ratio of the variance over the mean of the background-corrected spot-forming cells (SFC) replicates obtained under stimulation with different peptide pools (CMV, CEF). This report includes the longitudinal analysis of the ELISpot program cohort composed of 22 laboratories from 2011 to 2022 to assess whether the within-lab variability has improved over time. Random intercept models of the dispersion over time showed a significant decrease in overall dispersion from an average of approximately 1.8 in 2011 to approximately 1.25 in 2022. Out of the 21 sites, 16 sites (4 being statistically significant) had a negative trend for dispersion over time. Our finding of a reduction of overall within-lab variability demonstrates the need for and benefit of proficiency testing programs.

ADIPOPHILIN PEPTIDE (ADPH 129-137) IS NOT A TARGET ANTIGEN FOR CD8+ T-CELLS IN PATIENTS WITH OBESITY.

Context: In obesity, the infiltration of leukocytes into adipose tissue seems to play a key role in the development of inflammation and insulin resistance. Over-expression of adipophilin (ADPH) in adipose tissue, a protein which regulates lipid droplet structure and formation, has been reported in some studies. Objective: To investigate the role of ADPH 129-137 as a target for CD8+ T-cells in PBMCs of patients with obesity. Subjects and Methods: PBMCs were obtained from 9 non-diabetic obese patients and 11 healthy subjects expressing the HLA-A0201 molecule. The ELISPOT assay used to monitor the presence of IFN-γ producing CD8+ T-cells against a HLA class I-restricted epitope derived from Adipophilin (ADPH 129-137) and two control peptides: Flu MP58-66 and Melan-A27-35. Results: The outcomes showed no significant difference between patient group and healthy donors in response to ADPH 129-137. Conclusion: These results demonstrated that ADPH 129-137 peptide possibly does not act as an autoantigen in patients with obesity.

Enzyme-linked ImmunoSpot (ELISpot) assay to quantify peptide-specific IFN-γ production by splenocytes in a mouse tumor model after radiation therapy.

To develop new effective therapeutic strategies for cancer patients, there is a need for extensive and precise insights into the mechanisms involved in the immune response to anti-cancer treatments. The enzyme-linked immunospot (ELISpot) assay is a rapid and reproducible technique that allows the detection of cytokine-producing antigen-specific T cells at the single cell level. This protocol describes an interferon gamma (IFN-γ) ELISpot method for measuring antigen-specific murine CD8+ T cells that produce IFN-γ, a marker of their activation and cytotoxicity. Splenocytes from tumor-bearing mice treated with radiation therapy were used as source of CD8+ T cells and were stimulated with a tumor-derived peptide. This method was facilitated by a ready-to-use assay kit and provides a tool to analyze the specificity, intensity, and kinetics of specific CD8+ T cells.

The immunoglobulin M-degrading enzyme of Streptococcus suis (IdeSsuis) leads to long-lasting inhibition of the activation of porcine IgM-secreting B cells.

Streptococcus suis (S. suis) is one of the most important porcine pathogens, causing severe pathologies such as meningitis or polyarthritis. It is also a very successful colonizer of mucosal surfaces. The IgM-degrading enzyme of S. suis (IdeSsuis) specifically cleaves porcine IgM, which results in complement evasion. On the basis of our previous finding that IdeSsuis also cleaves the IgM B cell receptor in vitro, we verified IgM B cell receptor cleavage ex vivo in whole regional lymph nodes and investigated the working hypothesis that this IgM B cell receptor cleavage results in a long-lasting impaired B cell function. The number of IgM-secreting cells was determined via ELISpot analysis after porcine peripheral blood mononuclear cells had initially been treated with different recombinant S. suis proteins and subsequently stimulated with interleukin-2 and the toll-like receptor 7/8 ligand R848. Compared with treatment with medium or recombinant muramidase-released protein, treatment with rIdeSsuis but also with a cleavage-deficient variant led to a reduction in the number of IgM-secreting cells as well as the level of secreted IgM. Flow cytometry analysis confirmed that the IgM B cell receptor was cleaved only by rIdeSsuis, and the receptor recovered to pretreatment levels on day 2 after treatment. Flow cytometry analysis of B and T cells incubated with fluorescein-labelled recombinant proteins revealed that different rIdeSsuis variants bind specifically to B cells, most prominently the cleavage-deficient variant. Our results indicate that in vitro interference of rIdeSsuis with the IgM B cell receptor results in long-lasting impaired IgM secretion by B cells after toll-like receptor activation. Further studies are warranted to prove that the modulation of B cell function by IdeSsuis could play a role in vivo.

Oncolytic adenovirus MEM-288 encoding membrane-stable CD40L and IFNß induces an anti-tumor immune response in high grade serous ovarian cancer.

Single agent immune checkpoint inhibitors have been ineffective for patients with advanced stage and recurrent high grade serous ovarian cancer (HGSOC). Using pre-clinical models of HGSOC, we evaluated the anti-tumor and immune stimulatory effects of an oncolytic adenovirus, MEM-288. This conditionally replicative virus encodes a modified membrane stable CD40L and IFNß. We demonstrated this virus successfully infects HGSOC cell lines and primary human ascites samples in vitro. We evaluated the anti-tumor and immunostimulatory activity in vivo in immune competent mouse models. Intraperitoneal delivery of MEM-288 decreased ascites and solid tumor burden compared to controls, and treatment generated a systemic anti-tumor immune response. The tumor microenvironment had a higher proportion of anti-tumor macrophages and decreased markers of angiogenesis. MEM-288 is a promising immunotherapy agent in HGSOC, with further pre-clinical studies required to understand the mechanism of action in the peritoneal microenvironment and clinical activity in combination with other therapies.

Optimized Ex Vivo Fluorospot Assay to Measure Multifunctional HBV-Specific T Cell Frequencies in Chronic Hepatitis B Patients.

Virus-specific T cells are critical to mediating viral control; however, Hepatitis B virus (HBV)-specific T cells among chronic Hepatitis B (CHB) patients are functionally exhausted. The inability to consistently measure the ex vivo functionality of HBV-specific T cells has prevented meaningful analysis during antiviral events such as HBeAg seroconversion, hepatic flares, and HBsAg loss. We optimized the traditional IFN-γ ELISpot assay to measure total ex vivo HBV-specific T cell frequencies using CHB PBMCs stimulated with HBV overlapping peptide (OLP) pools. This was then further adapted to assess individual antigen specificity (core, envelop, polymerase, X) and multifunctional HBV-specific T cells using a 3-analyte FluoroSpot assay. This protocol encompasses two major components: (1) PBMC handling/stimulation and (2) assay plate preparation and spot development. By performing this assay, ex vivo CHB patient T cell responses could be assessed longitudinally during immunotherapy or other important clinical events.

Ex Vivo Fluorescent Labeling of HBV-Specific B Cells in Chronic Hepatitis B Patients.

Fluorescently conjugated antigen-bait systems have been extensively used to identify antigen-specific B cells and probe humoral immunity across different settings. Following this approach, we used HBV antigens to bind the B cell receptor (BCR), permitting antigen-specific B cell detection by flow cytometry. Fluorochromes can either be attached covalently via chemical conjugation to the antigen or attached non-covalently by biotinylating the antigen. Dual-staining antigen-baits (where an antigen is directly conjugated to two distinct fluorochromes) have now been used to identify HBsAg- and HBcAg-specific B cells with a high degree of reliability and specificity. This system can be used to detect and characterize cells ex vivo or adapted to isolate antigen-specific cells using fluorescence-activated cell sorting.

Quantification of RVFV Specific T Cell Responses in Mice Pre-immunized with Potential Vaccine Candidates.

Rift Valley fever (RVF) caused by Rift Valley fever virus (RVFV) is a major health concern for both domesticated animals and humans in certain endemic areas of Africa. With changing environmental conditions and identification of vectors capable of transmitting the virus, there is high risk of RVFV spreading into other parts of the world. Furthermore, unavailability of effective vaccines in the event of an outbreak can be a major challenge as witnessed recently in case of SARS-CoV2 pandemic. Hence, identifying potential vaccines and testing their protective efficacy in preclinical models before clinical testing is the absolute need of the hour. Here, we describe methods used to quantify virus-specific T cell responses in mice that were immunized with RVFV strains or antigens.

High glutamic acid decarboxylase antibody titers may be associated with a decline in ß-cell function over time and future insulin deficiency in latent autoimmune diabetes in adults.

AIMS: Latent autoimmune diabetes in adults (LADA) is characterized by positive islet-associated autoantibodies including glutamic acid decarboxylase antibody (GADA), and gradual decline in insulin secretion, progressing to insulin dependency. This cross-sectional study aimed to determine whether GADA by enzyme-linked immunosorbent assay (GADA-ELISA) titer of ≥180 U/mL could be associated with decline in ß-cell function in participants with LADA. METHODS: Sixty-three participants with LADA were recruited and an association between insulin secretion capacity and disease duration was investigated. Insulin peptide-specific inflammatory immunoreactivity was investigated to determine the disease's activity. RESULTS: There was a significant inverse correlation between disease duration and C-peptide index in participants with GADA-ELISA titer of ≥180 U/mL (Spearman's r (rs) = -0.516, p < 0.01). The positivity rate of insulin peptide-specific inflammatory immunoreactivity was significantly higher in those with ≥180 U/mL than in those with <180 U/mL (p < 0.05). In participants with human leukocyte antigen (HLA)-DRB1*04:05, a significant inverse correlation was observed between disease duration and C-peptide index in those with ≥180 U/mL (rs = -0.751, p < 0.01). CONCLUSIONS: GADA-ELISA titer of ≥180 U/mL, especially with HLA-DRB1*04:05, might reflect higher disease activity and may be associated with decline in ß-cell function over time and future insulin dependency in LADA.

Immune reconstitution and cidofovir administration rescue human adenovirus hepatitis after allogeneic hematopoietic cell transplantation.

Human adenovirus infection (HAdV) may be fatal in patients undergoing allogeneic hematopoietic cell transplantation (HCT). Cidofovir is effective in only a part of the post-HCT HAdV infection. Therefore, posttransplant immune reconstitution is important for HAdV clearance. We describe the detailed immune reconstitution and response of adenovirus-specific T cells in a patient with inborn errors of immunity who had disseminated HAdV infection with hepatitis post-HCT and was treated with cidofovir. Though the patient received cidofovir for only 19 days starting from Day 72 after HCT because of renal dysfunction, we observed T-cell reconstitution, a decrease in HAdV copy number, and amelioration of the symptoms of HAdV infection after Day 90. We initially observed expanded NK and CD8+CD45RO+ memory subsets and later gradual increase of naïve T cells eveloped after cessation of cidofovir treatment. An increase in adenovirus-specific IFN-γ secretion from 2 to 4 months after HCT was confirmed by ELISpot assay. The progression of immune reconstitution and cidofovir treatment are considered to have contributed to survival in this patient. Optimization of transplantation methods, prompt appropriate antiviral medication, and virus-specific T-cell therapy would be necessary as the better strategy for systemic HAdV infection.

Strategies to determine positive anti-SARS-CoV-2 memory T lymphocyte response during the evolution of an epidemic.

During SARS-CoV-2 pandemic, the assessment of immune protection of people at risk of severe infection was an important goal. The appearance of VOCs (Variant of Concern) highlighted the limits of evaluating immune protection through the humoral response. While the humoral response partly loses its neutralizing activity, the anti-SARS-CoV-2 memory T cell response strongly cross protects against VOCs becoming an indispensable tool to assess immune protection. We compared two techniques available in laboratory to evaluate anti-SARS-CoV-2 memory T cell response in a cohort of infected or vaccinated patients with different levels of risk to develop a severe disease: the ELISpot assay and the T-Cell Lymphocyte Proliferation Assay respectively exploring IFNγ production and cell proliferation. We showed that the ELISpot assay detected more anti-Spike memory T cell response than the Lymphocyte Proliferation Assay. We next observed that the use of two different suppliers as antigenic source in the ELISpot assay did not affect the detection of anti-Spike memory T cell response. Finally, we explored a new approach for defining the positivity threshold, using unsupervised mixed Gaussian modeling, challenging the traditional ROC curve used by the supplier. That will be helpful in endemic situation where it could be difficult to recruit "negative" patients.

Multiparametric analysis of the specific immune response against SARS-CoV-2.

BACKGROUND: SARS-CoV-2, which causes COVID-19, has killed more than 7 million people worldwide. Understanding the development of postinfectious and postvaccination immune responses is necessary for effective treatment and the introduction of appropriate antipandemic measures. OBJECTIVES: We analysed humoral and cell-mediated anti-SARS-CoV-2 immune responses to spike (S), nucleocapsid (N), membrane (M), and open reading frame (O) proteins in individuals collected up to 1.5 years after COVID-19 onset and evaluated immune memory. METHODS: Peripheral blood mononuclear cells and serum were collected from patients after COVID-19. Sampling was performed in two rounds: 3-6 months after infection and after another year. Most of the patients were vaccinated between samplings. SARS-CoV-2-seronegative donors served as controls. ELISpot assays were used to detect SARS-CoV-2-specific T and B cells using peptide pools (S, NMO) or recombinant proteins (rS, rN), respectively. A CEF peptide pool consisting of selected viral epitopes was applied to assess the antiviral T-cell response. SARS-CoV-2-specific antibodies were detected via ELISA and a surrogate virus neutralisation assay. RESULTS: We confirmed that SARS-CoV-2 infection induces the establishment of long-term memory IgG+ B cells and memory T cells. We also found that vaccination enhanced the levels of anti-S memory B and T cells. Multivariate comparison also revealed the benefit of repeated vaccination. Interestingly, the T-cell response to CEF was lower in patients than in controls. CONCLUSION: This study supports the importance of repeated vaccination for enhancing immunity and suggests a possible long-term perturbation of the overall antiviral immune response caused by SARS-CoV-2 infection.

Epitope mapping and a candidate vaccine design from canine distemper virus.

Background: Canine distemper (CD) is a worldwide spread disease that has been described in 12 families of mammals, especially in the Carnivora order, being better studied in domestic canines where vaccination represents the best means of control. CD is controlled by vaccination, but many cases of the disease still occur in vaccinated animals. Aim: The aim of this work was to study antigen-specific epitopes that can subsidize the development of a new vaccine approach. Methods: Mapping of T cell reactive epitopes for CD virus (CDV) was carried out through enzyme-linked immunospot assays using 119 overlapped synthetic peptides from the viral hemagglutinin protein, grouped in 22 pools forming a matrix to test the immune response of 32 animals. Results: Evaluations using the criteria established to identify reactive pools, demonstrated that 26 animals presented at least one reactive pool, that one pool was not reactive to any animal, and six pools were the most frequent among the reactive peptides. The crisscrossing of the most reactive pools in the matrix revealed nine peptides considered potential candidate epitopes for T cell stimulation against the CDV and those were used to design an in-silico protein, containing also predicted epitopes for B cell stimulation, and further analyzed using immune epitope databases to ensure protein quality and stability. Conclusion: The final in silico optimized protein presents characteristics that qualify it to be used to develop a new prototype epitope-based anti-CDV vaccine.

Virus-specific antibody secreting cells reside in the peritoneal cavity and systemic immune sites of Atlantic salmon (Salmo salar) challenged intraperitoneally with salmonid alphavirus.

The development and persistence of antibody secreting cells (ASC) after antigenic challenge remain inadequately understood in teleosts. In this study, intraperitoneal (ip) injection of Atlantic salmon (Salmo salar) with salmonid alphavirus (WtSAV3) increased the total ASC response, peaking 3-6 weeks post injection (wpi) locally in the peritoneal cavity (PerC) and in systemic lymphoid tissues, while at 13 wpi the response was only elevated in PerC. At the same time point a specific ASC response was induced by WtSAV3 in PerC and systemic tissues, with the highest frequency in PerC, suggesting a local role. Inactivated SAV (InSAV1) induced comparatively lower ASC responses in all sites, and specific serum antibodies were only induced by WtSAV3 and not by InSAV1. An InSAV1 boost did not increase these responses. Expression of immune marker genes implies a role for PerC adipose tissue in the PerC immune response. Overall, the study suggests the Atlantic salmon PerC as a secondary immune site and an ASC survival niche.